Immunoprotective influenza antigen and its use in vaccination

ABSTRACT

The present invention relates to an influenza antigen, comprising a fusion product of at least the extracellular part of a conserved influenza membrane protein or a functional fragment thereof and a presenting carrier, which may be a presenting (poly)peptide or a non-peptidic structure, such as glycans, peptide mimetics, synthetic polymers. The invention further relates to a vaccine against influenza, comprising at least an antigen of the invention, optionally in the presence of one or more excipients. The invention also relates to use of the antigen, a method for preparing the antigen and acceptor cells expressing the antigen.

CROSS REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. §§120 and 365(c), this application claims benefitof parental application Ser. No. 09/498,046, filed Feb. 4, 2000, nowU.S. Pat. No. 7,731,972, that claimed the benefit of internationalapplication PCT/EP98/05106, filed Aug. 5, 1998, designating the UnitedStates, which application claimed the benefit of prior Europeanapplication EP 97202434.3, filed Aug. 5, 1997.

FIELD OF THE INVENTION

The present invention relates to new immunoprotective influenzaantigens, which are non-existent in nature. The invention furtherrelates to the use of the antigens for vaccination and to vaccinescontaining them, as well as to methods for preparing the antigens.

BACKGROUND OF THE INVENTION

Influenza is caused by an RNA virus of the myxovirus group. Influenzaviruses can be classified into three types (A, B and C), based onantigenic differences in the nucleoprotein and the matrix protein. TypeA and B influenza viruses each contain 8 RNA segments, while type C onlyhas 7 RNA segments. Influenza A is most important and is very pathogenicfor man, as well as for animals, for example pigs and horses. Type Binfluenza causes disease in humans. Influenza C is less severe and wasisolated from humans and pigs. The virus is transmitted through the air,mainly in droplets expelled during coughing and sneezing. The influenzaviruses cause an infection of the respiratory tract, that is usuallyaccompanied with coughing, high fever and myalgia. Although an influenzainfection does not often lead to the death of the infected individual,the morbidity can be severe. As a consequence thereof influenzaepidemics may lead to substantial economic loss. Furthermore, influenzainfection can be more dangerous for certain groups of individuals, suchas those having suffered from a heart attack, CARA patients or elderly.A vaccine against influenza is therefore highly desirable.

The influenza A virus contains in its membrane two highly immunogenic,but very variable proteins, the haemagglutinin and the neuraminidase.Due to the variability of these two proteins a broad spectrum longlasting vaccine against influenza A has so far not been developed. Theinfluenza vaccine commonly used, has to be adapted almost every year tofollow the antigenic drift of the virus. In these circumstances thevaccine can protect about 80% of the immunized persons. When moredrastic changes occur in the virus, known as antigenic shift, thevaccine is no longer protective.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to provide a newimmunoprotective antigen for use in vaccines which is not based on therapidly changing haemagglutinin and/or neuraminidase and which thereforelacks the disadvantages of these known antigens and vaccines basedthereon.

In the research that led to the present invention it was found that wellconserved membrane proteins of influenza other than haemagglutinin andneuraminidase can be used for eliciting protection. Particularly usefulfor this approach is the membrane protein M2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Construction of pATIPM2 ml.

E1 and E2=first and second exon of the influenza M2 protein

M2e=extracellular part of the M2 protein,

M2t=transmembrane part; and

M2c=cytoplasmic tail.

Bold line=vector

(a) removal of the intron out of the m2 gene SEQ ID NO: 59, E1 top andbottom [SEQ ID NOs:6, 7]

(b) introduction of a BclI site between the extracellular part and thetransmembrane domain of the M2 protein, mutator [SEQ ID NO:8]

(c) nucleotide (SEQ ID NO:9) and amino acid sequence (SEQ ID NO:10) ofthe extracellular part of the M2 protein of A/PR/8/34, nucleotidesequence and amino acid sequence [SEQ ID NOs:9, 10].

FIG. 2: Construction of pIPM2hB2Mm2s2.

Ori=origin of replication,

Cat=chloramphenicol acetyltransferase,

Bla=β-lactamase,

Lpp=lipoprotein,

hB2M=human β₂-microglobulin,

ompa-ss=signal sequence of the outer membrane protein A of E. coli,

ssDNA=single-stranded DNA,

M2e=extracellular part of the M2 protein.

(a) Construction flow scheme, FIG. 2 a 2 mutator sequences (top andbottom) [SEQ ID NOs:11 and 12], FIG. 2 a 3 linkers (top and bottom) [SEQID NOs:13 and 14], FIG. 2 a 3 mutator [SEQ ID NO:15]

(b) Details of key sequences that include paired oligonucleotide andencoded amino acid sequences of SEQ ID NOs:44-51.

FIG. 3: Construction of pPLcIPM2HBcm.

Ori=origin of replication,

Cat=chloramphenicol acetyltransferase

Bla=β-lactamase,

HBc=hepatitis B core,

ssDNA=single-stranded DNA,

M2e=extracellular part of the M2 protein.

(a) Plasmid construction flow scheme, FIG. 3 a 2 mutator [SEQ ID NO:16],FIG. 3 a 4 mutator [SEQ ID NO:17], FIG. 3 a 4 selection [SEQ ID NO:18],

(b) Sequence around the introduced BamHI restriction site in thehepatitis B core gene that includes paired oligonucleotide and encodedamino acid sequences of SEQ ID NOs:52-55 (FIG. 3 b),

(c) Details of key sequences that include paired oligonucleotide andencoded amino acid sequences of SEQ ID NOs:56-61 (FIG. 3 c).

FIG. 4: Analysis of the soluble fraction, corresponding to 150 μloriginal culture, of strain MC1061[pcI857] containing the plasmidspPLc245 (control), pPLcA1 (expression of HBc) or pPLcIPM2HBcm(expression of IPM2HBcm) respectively, on a SDS 12.5% PAGE. After theelectrophoresis the gel was stained with Coomassie brilliant blue.

MW=molecular weight marker,

NI=not induced culture,

I=induced culture.

FIG. 5: Analysis of the soluble fraction, corresponding to 150 μloriginal culture, of strain MC1061[pcI857] transformed with pPLc245(control), pPLcA1 (expression of HBc) or pPLcIPM2HBcm (expression ofIPM2HBcm) respectively, as in FIG. 4. After electrophoresis, therelevant proteins were revealed by a Western blotting experiment.Detection with (A) a monoclonal antibody against HBc and (B) amonoclonal antibody specific for the extracellular part of the M2protein.

MW=molecular weight marker,

NI=not induced culture,

I=induced culture.

FIG. 6: Sequence of the amino terminus of the M2 protein compared to theamino terminus of IPM2HBcm as an oligonucleotide, its translated aminoacid residue sequence (SEQ ID NOs: 19 and 20) the amino acid residuesequence of the amino terminus of the fusion protein IPM2HBcm (SEQ IDNO: 21), as experimentally determined. Sequence of A/Udorn/72 SEQ ID NO:62, (Lamb and Zebedee, 1985).

FIG. 7: Soluble fractions of strain MC1061 [pcI857] transformed withpPLc245 (control), pPLcA1 (expression of HBc) or pPLcIPM2HBcm(expression of IPM2HBcm), respectively, analyzed in a native state bymeans of a dot blot. Detection with (A) a monoclonal antibody againstHBc and (B) a monoclonal antibody specific for the extracellular part ofthe M2 protein.

NI=not induced culture,

I=induced culture.

FIG. 8: Overview of (A1) rectal temperature, (A2) weight and (B)survival of the mice vaccinated with IPM2HBcm after a lethal challengewith 5 LD₅₀ m.a. A/PR/8/34. The statistical significance was calculatedby the Fisher's exact test. Mice immunized with different doses ofantigen were compared to the control group. The following results wereobtained: for 50 μg IPM2HBcm p<0.001; for 10 μg p<0.005 and for the 5 μgdose p<0.05. FIG. 8C shows the survival of the mice vaccinatedintraperitoneally with IPM2HBcm, and IM2HBcm, respectively, after alethal challenge with 30 HAU X-47. FIG. 8D shows the survival of themice vaccinated intranasally with IPM2HBcm, and IM2HBcm, respectively,after a lethal challenge with HAU X-47.

FIG. 9: Analysis of the serum samples of the four set ups reported inFIG. 8. The pre-immune serum (a), the serum taken after the first (b),after the second (c) and after the third (d) immunization and the serumtaken after challenge (e) were initially diluted 1/50. The consecutivedilution steps were 1/3. The plotted absorbance is a corrected valueobtained as described in Results, Analysis of the serum samples.

FIG. 10: Construction of pPLcIM2HBcm including the site-directedmutagenesis mutator oligonucleotide (SEQ ID NO: 22) and selectionoligonucleotide (SEQ ID NO: 23),

ori=origin of replication,

cat=chloramphenicol acetyltransferase,

bla=β-lactamase,

M2e=extracellular part of the M2 protein,

HBc=hepatitis B core.

FIG. 11: Analysis of the soluble fraction, containing 5 μg HBc orI(P)M2HBcm (as determined in an ELISA (see Materials and Methods)), ofstrain MC1061 [pcI857] containing respectively the plasmids pPLc245(control), pPLcA1 (expression of HBc), pPLcIPM2HBcm (expression of thefusion protein IPM2HBcm with the extracellular part of the M2 proteinderived from A/PR/8/34) or PPLcIM2HBcm (expression of IM2HBcm,containing the more universal M2 sequence) on a SDS 12.5% PAGE-gel.

MW=molecular weight marker,

NI=not induced,

I=induced culture.

FIG. 12: Analysis of the soluble fraction, containing 2.5 μg HBc orI(P)M2HBcm (as determined in an ELISA (see Materials and Methods)), ofstrain MC1061 [pcI857] containing respectively the plasmids pPLc245(control), pPLcA1 (expression of HBc), pPLcIPM2HBcm (expression ofIPM2HBcm) or pPLcIM2HBcm (expression of IM2HBcm) on a Western blot (seeMaterials and Methods). Detection with (A) a monoclonal antibodydirected against HBc and (B) a monoclonal antibody specific for theextracellular part of the M2 protein.

MW=molecular weight marker,

NI=not induced,

I=induced culture.

FIG. 13: Overview of the oligonucleotides (SEQ ID NOs: 24-27) used forPCR amplification of HBc and i(p)_(m2)HBc. ‘s’ or ‘a’ following the nameof the oligonucleotide stands for the use of these primers in the sense(s) or anti-sense (a) orientation. The boxed sequence indicates thechanged Leu codons.

FIG. 14: Overview of the construction of hbc and m2hbc fusions invectors for L. lactis

ori=origin of replication for E. coli E. coli,

ori(+)=origin of replication for L. lactis

ermA and ermM=erythromycin resistance genes,

P1=L. lactis promoter,

Bla=β-lactamase,

HBc=hepatitis B core,

M2e=extracellular part of the M2 protein,

usp45-ss=signal sequence of usp45,

mIL2=murine interleukin 2 and

mIL6=murine interleukin 6.

FIG. 15: Analysis of the expression of Hepatitis B core (HBc) and M2-HBcfusion proteins in a Western blot. An equivalent of 10⁹ L. lactisbacteria of strain MG1363 containing respectively pTREX1 (control),pT1HBc, pT1HBcIL2, pT1HBcIL6 (expression of HBc alone or in combinationwith mIL2 or mIL6, respectively), pT1PM2HBc, pT1PM2HBcIL2, pT1PM2HBcIL6(expression of IPM2HBcm alone or in combination with mIL2 or mIL6,respectively), pT1M2HBc, pT1M2HBcIL2, pT1M2HBcIL6 (expression of IM2HBcmalone or in combination with mIL2 or mIL6, respectively), was analyzedin a SDS 12.5% PAGE-gel. The first antibody, p-anti-HBc (DakoCorporation, Carpinteria, Calif., USA) was diluted 5000 times. The boundantibodies were detected with a 1/2000 dilution of the polyclonalanti-rabbit IgG labeled with alkaline phosphatase (SouthernBiotechnology Associates, Birmingham, Ala., USA). I(P)M2HBc stands foreither IPM2HBcm or IM2HBcm.

MW=molecular weight marker,

C=control and

−=expression of the antigen alone.

FIG. 16: Analysis of the expression of M2-HBc fusion proteins in aWestern blot. An equivalent of 2 to 3×10⁹ L. lactis bacteria of strainMG1363 containing respectively pT1HBc (control), pT1PM2HBc, pT1PM2LHBc(expression of IPM2HBcm), pT1M2HBc, pT1M2LHBc (expression of IM2HBcm),was separated on a SDS 12.5% PAGE-gel. The fusion proteins were detectedwith an IgG fraction of a polyclonal mouse anti-M2e antibody (seeMaterials and Methods). The bound antibodies were detected with a 1/2000dilution of the alkaline phosphatase conjugated polyclonal anti-mouseIgG (γ-chain specific) (Southern Biotechnology Associates, Birmingham,Ala., USA).

MW=molecular weight marker,

C=control,

E=leucine codons optimal for use in E. coli and

L=leucine codons optimal for use in L. lactis.

These are the plasmids pT1PM2LHBc and pT1M2LHBc, respectively. I(P)M2HBcstands for either IPM2HBcm or IM2HBcm.

FIG. 17: Overview of the oligonucleotides (SEQ ID NOs: 28-31) used forPCR amplification of the extracellular part of the M2 protein and C3d.

‘s’ or ‘a’ following the code name of the oligonucleotide stands for theuse of these primers in the sense (s) or anti-sense (a) orientation. Theboxed region indicates the changed Leu codons.

FIG. 18: Overview of the construction of m2c3d3 fusions in L. lactis.

ori=origin of replication for E. coli.

ori(+)=origin of replication for L. lactis,

ermA and ermM=erythromycin resistance genes,

P1=L. lactis promoter,

Bla=β-lactamase,

M2e=extracellular part of the M2 protein,

usp45-ss=signal sequence of usp45,

spaX=anchor sequence derived from Staphylococcus aureus protein A,

C3d=complement protein 3 fragment d, and

mIL6=murine interleukin 6.

FIG. 19: Overview of the oligonucleotides (SEQ ID NOs: 32-34) used forPCR amplification of TTFC and m2TTFC. ‘s’ or ‘a’ following the name ofthe oligonucleotide stands for the use of these primers in the sense (s)or anti-sense (a) orientation. The boxed region indicates the changedLeu codons.

FIG. 20: Overview of the construction of m2TTFC in vectors for L.lactis.

ori=origin of replication for E. coli,

ori(+)=origin of replication for L. lactis

ermM and ermμ=erythromycin resistance genes,

P1=L. lactis promoter,

Bla=β-lactamase,

TTFC=tetanus toxin fragment C,

M2e=extracellular part of the M2 protein,

usp45-ss=signal sequence of usp45,

mIL2=murine interleukin 2, and

mIL6=murine interleukin 6.

FIG. 21: Analysis of the expression of IPM2TTFC fusion protein in aWestern blot. An equivalent of 10⁹ L. lactis bacteria of strain MG1363containing respectively pT1TT (control), pT1PM2LTT (expression ofIPM2TT), pT1PM2LTTIL2 (expression of IPM2TT in combination with mIL2) orpT1PM2LTTIL6 (expression of IPM2TT in combination with mIL6), wasanalyzed in a SDS 10% PAGE-gel. The first antibody, an IgG fraction of apolyclonal mouse anti-M2e antibody (see Materials and Methods) wasdiluted 2500 times. The bound antibodies were detected with a 1/2000dilution of the polyclonal anti-mouse IgG labeled with horseradishperoxidase (Southern Biotechnology Associates, Birmingham, Ala., USA).30 mg 4-chloro-1-naphthol (Sigma Chemical Co., St. Louis, Mo., USA), wassolublized in 10 ml methanol.) Afterwards 40 ml PBS, pH 7.4 and 150μl_H₂O₂ was added.

MW=molecular weight marker,

−=expression of the antigen alone,

mIL2=expression of the antigen in combination with

mIL2, mIL6=expression of the antigen in combination with mIL6.

FIG. 22: Primer set (SEQ ID NOs: 35 and 36) used for PCR amplificationof the secretion signal of the gp67 baculovirus protein.

FIG. 23: Primer set (SEQ ID NOs: 37 and 38)used for PCR amplification ofthe extracellular part of the M2 protein during construction of thesgpM2C3d3 fusion.

FIG. 24: Construction of the baculovirus transfer vector pACsgpM2C3d3.

Bla=β-lactamase,

bold grey line=baculovirus homology region,

C3d=complement protein 3 fragment d,

M2e=extracellular part of the M2 protein (SEQ ID NO: 64),

ori=origin of replication,

phP=baculovirus polyhedrin promoter, and

sgp67=secretion signal of the gp67 baculovirus protein.

FIG. 25: Detail of nucleotide and amino acid key sequences of thesgpM2C3d3 fusion.

C3d=complement protein 3 fragment d (SEQ ID NOs: 68 and 67),

M2e=extracellular part of the M2 protein (SEQ ID NOs: 66 and 65), and

sgp67=secretion signal of the gp67 baculovirus protein.

FIG. 26: Analysis of recombinant AcNPV/sgpM2C3d3 baculovirus by PCRamplification of the polyhedrin locus (primers TTTACTGTTTTCGTAACAGTTTTGand CAACAACGCACAGAATCTAG SEQ ID NOs: 4 and 5). Control reactions wereperformed with the parental transfer vector pACsgpM2C3d3 and with wildtype AcNPV baculovirus.

M=DNA size markers.

FIG. 27: Expression of secreted M2C3d3 by sf9 insect cells infected withrecombinant AcNPV/sgpM2C3d3 baculovirus as demonstrated by Westernanalysis (10% PAGE-gel) of harvested supernatant. Supernatant from mockinfected cells or obtained after infection with wild type AcNPVbaculovirus are included as a control.

MW=molecular weight markers.

FIG. 28: Overview of the survival of mice after a lethal challenge with5 LD₅₀ m.a. X47. M1ce vaccinated with 3×10 μg IM2HBcm are compared withpassively immunized mice (P).

FIG. 29: Overview of the DNA vaccination constructs including theoligonucleotides of SEQ ID NOs: 39-43.

RT=reverse transcriptase

PCMV=cytomegalovirus promoter

Bla=β-lactamase

Npt=neomycin resistance.

FIG. 30 Expression in HEKT cells analyzed on a Western blot. The firstantibody (paM2 (see

Materials and Methods)) was diluted 2000 times. The bound anti-M2antibodies were detected with an alkaline phosphatase labeled anti-mouseIgG.

MW=molecular weight marker

M2=M2 protein expressed in insect cells

1=pcDNA3

2=pCIM2

3=pCIM2HBcm

4=pCIP3M2HBcm.

FIG. 31 Antibody response against the M2 protein analyzed in an ELISA.

A. Microtiterplates were coated with periplasm containing hB2M orIPM2hB2M respectively (see Materials and Methods).

B. Microtiterplates coated with M2 protein expressed in insect cells(see Materials and Methods).

DETAILED DESCRIPTION OF THE INVENTION

M2 mRNA is encoded by RNA segment 7 of the influenza A virus. It isencoded by a spliced mRNA (Lamb et al., 1981). Like the haemagglutininand the neuraminidase, the M2 protein is an integral membrane protein ofthe influenza A virus. But the protein is much smaller, only 97 aminoacids long. 24 Amino acids at the amino terminus are exposed outside themembrane surface, 19 amino acids span the lipid bilayer, while theremaining 54 residues are located on the cytoplasmic side of themembrane (Lamb et al., 1985).

The M2 protein is abundantly expressed at the cell surface of influenzaA infected cells (Lamb et al., 1985). The protein is also found in themembrane of the virus particle itself, but in much smaller quantities,14 to 68 molecules of M2 per virion (Zebedee and Lamb, 1988). The M2protein is posttranslationally modified by the addition of a palmiticacid on cysteine at position 50 (Sugrue et al., 1990).

The M2 protein is a homotetramer composed of two disulfide-linkeddimers, which are held together by noncovalent interactions (Sugrue andHay, 1991). By site-directed mutagenesis, Holsinger and Lamb (1991)demonstrated that the cysteine residue at position 17 and 19 areinvolved in disulfide bridge formation. Only cysteine at position 17 ispresent in all viruses analysed, therefore it seems likely that this isthe most important residue. In the virus strains where cysteine 19 isalso present, it is not known whether a second disulfide bridge isformed in the same dimer (linked by Cys 17-Cys 17) or with the other.

By aligning the sequences of M2 proteins, isolated from different humanstrains of influenza A virus, a striking conservation of theextracellular part of the M2 protein, became evident (table 1). Sincethe first human influenza A strain isolated in 1933, A/WS/33 (H1N1),until the most recently sequenced virus A/Guangdong/39/89 (H3N2), noamino acid change has been observed in the extracellular domain of theM2 protein. Two virus strains do not fit in this conserved pattern,A/PR/8/34 (H1N1), which shows one amino acid change and A/FortMonmouth/1/47 (H1N1), which shows three amino acid differences. Thesetwo strains probably represent side branches in the evolutionary tree.

Table 1 gives an overview of the amino acid sequences of theextracellular domain of the influenza A M2 protein of the virus strainsA/WSN/33 (Markushin et al. (1988)), A/PR/8/34 (Allen et al. (1980),Winter and Fields (1980)), A/WS/33, A/Fort Warren/1/50, A/Singapore/1/57and A/Port Chalmers/1/73 (all described by Zebedee and Lamb (1989)),A/Udorn/72 (Lamb and Lai (1981)), A/Leningrad/134/57 (Klimov et al.(1992)), A/Ann Arbor/6/60 (Cox et al. (1988)), A/Bangkok/1/79 (Ortin etal. (1983)), A/New York/83 (Belshe et al. (1988)), A/Fort Monmouth/1/47(EMBL U02084), A/USSR/90/77 (EMBL X53029) and A/Guangdong/39/89 (EMBL L18999).

TABLE 1 Amino acid sequence of the extracellular domain of the M2protein2  3  4  5  6  7  8  9  10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Strain SEQ ID NO A/WS/33 (H1N1)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/WSN/33 (H1N1)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/PR/8/34 (H1N1)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnGlySerSerAsp 2A/Fort Monmouth1/47 (H1N1)SerLeuLeuThrGluValGluThrProThrLysAsnGluTrpGluCysArgCysAsnAspSerSerAsp 3A/Fort Warren/1/50 (H1N1)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/USSR/90/77 (H1N1)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/Singapore/1/57 (H2N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/Leningrad/134/57 (H2N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/Ann Arbor/6/60 (H2N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/Udorn/73 (H3N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/Port Chalmers/1/73 (H3N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/Bangkok/1/79 (H3N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/NY/83 (H3N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1A/Guangdong/39/89 (H3N2)SerLeuLeuThrGluValGluThrProIleArgAsnGluTrpGlyCysArgCysAsnAspSerSerAsp 1

It was anticipated by the present inventors that the conserved characterof this type of membrane proteins could make them a good candidate forvaccine development. In principle the protective capacity of anti-M2antibodies is already known. Experimental data demonstrated that amonoclonal antibody directed against the extracellular part of the M2protein (14C2) can diminish the spread, although the infectivity of thevirus in vitro was not reduced (Zebedee and Lamb, 1988). Furthermore itwas demonstrated that passively administered monoclonal antibody (14C2)could inhibit viral replication in the lungs of mice (Treanor et al.,1990). Both approaches rely on the administration of anti-M2 antibodies.However, the passive administration of monoclonal antibodies ispreferably avoided because of the immunogenicity of heterologousimmunoglobulins which, upon repeated administration, can lead to theclearing of the antibodies from the body and thus to a reduction of theefficacy of the treatment. Even homologous antibodies can elicitanti-idiotype antibodies. Furthermore, it was found that humans infectedwith the virus do have anti-M2 antibodies but these do not protectagainst infection, (either their concentration or their nature are notsufficient to confer efficacy). This makes it unlikely that passiveadministration of anti-M2 antibodies is suitable for use in humans. Italso teaches away from trying to develop vaccines for humans based onthis antigen.

Recently, protection of mice against an infection with homologous orheterologous virus was described (Slepushkin et al., 1995). Theseauthors used a formulation of incomplete Freund's adjuvant and amembrane extract of Sf9 cells expressing the complete M2 protein forimmunizations. However, this approach is also not suitable forvaccination of humans because it relies on the use of Freund's adjuvantwhich is prohibited in humans.

In summary, use of antibodies for providing protection against influenzais preferably to be avoided. Moreover, it is unlikely that prophylactictreatment with antibodies will be effective in humans. Immunization withcomplete M2 protein in humans as described is not possible because itrelies on incomplete Freund's adjuvant which cannot be used in humans,and is counter-indicated in higher animals.

It is thus the object of the invention to provide for an alternativeinfluenza antigen that is sufficiently immunoprotective and is notdependent on Freund's adjuvant to find a use in humans.

According to the invention it has now been found that it is possible toprepare such a novel antigen that does not exist in nature. For this theextracellular part of a conserved influenza membrane protein or afunctional fragment thereof is fused to a presenting carrier, forexample a (poly)peptide. The conserved influenza membrane protein is forexample the well conserved, extracellular part of the M2 protein. Themembrane protein is preferably genetically fused to a presenting(poly)peptide as the presenting carrier, which (poly)peptide stabilisesthe extracellular part and surprisingly potentiates the immunogenicityof the fusion product thus obtained. It is thought that the presenting(poly)peptide brings the extracellular part into its wild typestructure, thus presenting the antigen in a form that is also found onthe virus and on the infected cells.

A ‘functional fragment of the conserved influenza membrane protein’ is afragment that is capable of eliciting a statistically significant higherimmunoprotection when administered in an immunoprotective dose to testmembers of a species than is found in control members of the samespecies not receiving the functional fragment.

In one embodiment of the invention the 23 amino acid extracellular partof the M2 protein is fused to the amino terminus of the human HepatitisB virus core protein. In this way the wild type structure of the M2protein in viral particles and on infected cells is mimicked, where thefree N-terminus extends in the extracellular environment.

Alternative presenting (poly)peptides are multiple C3d domains (Dempseyet al., 1996), tetanus toxin fragment C or yeast Ty particles.‘Presenting (poly)peptides’ are intended to encompass every stretch ofamino acid(s) that can present the extracellular part, in asubstantially wild type form, towards the environment.

Alternatively, the presenting carrier can be a non-peptidic structure,such as glycans, peptide mimetics, synthetic polymers, etc.

After expression of the novel antigen in a suitable acceptor cell, itcan be used either as such (depending on the acceptor cell), as part ofa membrane fragment or in isolated form.

The term ‘presenting carrier’ is used to indicate all types ofpresenting molecule, both (poly)peptides and others.

It will be clear for the person skilled in the art that a geneconstruct, comprising the coding information for the antigen and thepresenting (poly)peptide, can not only be used to prepare the newantigen, as described above, but that it can also be used, optionally inthe presence of suitable transcription and/or translation regulatorysequences, in a DNA vaccine, or in vaccinia based vaccine constructions.

A presenting (poly)peptide can be incorporated into the fusion productin a single copy or in multiple copies. The third complement proteinfragment d (C3d) is preferably used in more copies, preferably 3.

In a preferred embodiment of the invention the fusion product furthermay comprise an additional peptide at an appropriate internal site(Schödel et al., 1992) or C-terminal (Borisova et al., 1989). Thisadditional peptide is intended to further increase the protectivecapacity of the antigen, and may for example be a T helper cell epitopeor a cytotoxic T cell epitope.

The antigen of the invention is obtainable by preparing a gene constructcomprising a coding sequence for at least the extracellular part of aconserved influenza membrane protein or a functional fragment thereofand optionally the coding sequence for a presenting (poly)peptideoperably linked thereto, optionally in the presence of suitabletranscription and/or translation and/or secretion regulatory sequences,bringing this gene construct in a suitable acceptor cell, effectingexpression of the gene construct in the acceptor cell and optionallyisolating the antigen from the acceptor cell or its culture medium.

The presence of the transcription and/or translation and/or secretionregulatory sequences depends on whether the gene is to be integratedinto a vector or the genome of the acceptor cell at a position alreadyproviding these signals.

The coding sequence for a presenting (poly)peptide is only present whenthe fusion product is a fusion between the antigen and a peptidicstructure and if it is desirable to directly link the two structures inthe DNA construct. In all other instances, the presenting carrier may beadded to the antigen in a different manner.

The suitable acceptor cell can be selected from E. coli, Lactococcuslactis, Lactobacillus plantarum, yeast (e.g. Pichia pastoris). In thecase of L. lactis the antigen need not be isolated but the engineeredbacteria can be used directly for intranasal or oral use.

The invention further relates to vaccines that comprise at least theantigen of the invention. This antigen can be in isolated form or beingpart of a membrane fragment or being expressed on the acceptor cell. Theantigen of the invention can be used together with suitable excipients.The person skilled in the art of vaccine design will be capable ofselecting suitable excipients. Guidance may for example be found inMethods in molecular medicine: Vaccine Protocols (1996). Eds. Robinson,A., Farrar, G. H. and Wiblin, C. N. Humana Press, Totowa, N.J., USA.

The antigens of the invention may be used alone or in combination withone or more other influenza antigens, such as neuraminidase,haemagglutinin or native M2.

Furthermore, the invention relates to the use of the antigens in thepreparation of a vaccine against influenza. The vaccines can be directvaccines, i.e. vaccines containing the fusion products or indirect DNAvaccines. The latter are vaccines, comprising the fusion cDNA under theregulation of a eukaryotic promoter that can function in the recipient.The actual antigen is then produced in the recipient of the vaccine.

The vaccines of the invention are intended both for use in humans and inanimals, for example pigs and horses of which it is known that they areinfected by influenza A.

A similar approach as described here for preparing novel fusion antigensof influenza A can be adopted to prepare similar fusion antigens andvaccines containing the fusion antigens or DNA encoding the fusionantigens for influenza B and C.

The invention also relates to a method of preparing the antigens,comprising the steps of:

a) preparing a gene construct comprising a coding sequence for at leastthe extracellular part of a conserved influenza membrane protein or afunctional fragment thereof and at least one coding sequence for apresenting (poly)peptide operably linked thereto, optionally in thepresence of suitable transcription and/or translation and/or secretionregulatory sequences,

b) bringing this gene construct in a suitable acceptor cell,

c) effecting expression of the gene construct in the acceptor cell, and

d) optionally isolating the antigen from the acceptor cell or itsculture medium.

The invention will be further illustrated by the following example, thatis in no way intended to limit the invention. The example extensivelydescribes the preparation of fusion proteins of M2 with variouspresenting (poly)peptides and the use thereof in immunization. Insteadof M2 and the presenting carriers described here, the skilled personwill be capable of choosing another conserved influenza membrane proteinand other presenting carriers.

The following abbreviations will be used:

-   1 LD₅₀:the viral challenge required to kill half of the population    of infected mice-   BCIP: 5-bromo-4-chloro-3-indolylphosphate-   bp: base pair(s)-   CIP: calf intestine phosphatase-   C3d: complement protein 3 fragment d-   DEA: diethylamine-   HAU: haemagglutination units-   hB2M: human β2-microglobulin-   HBc: Hepatitis B core protein-   IM2HBcm: universal influenza A M2 protein fragment fused to HBc-   IPM2hB2Mm: influenza A M2 protein fragment (from A/PR/8/34) fused to    hB2M-   IPM2HBc: influenza A M2 protein fragment (from A/PR/8/34), fused to    HBc, containing four additional amino acids between the first    methionine and the start of the extracellular part of the M2 protein-   IPM2HBcm: influenza A M2 protein fragment (from A/PR/8/34) fused to    HBc-   IPTG: isopropyl-β-D-thiogalactoside-   m.a.: mouse adapted-   M2C3d3: universal influenza M2 fragment fused to three copies of C3d-   cM2C3d3: cytoplasmic form of M2C3d3-   sM2C3d3: secreted form of M2C3d3-   sM2C3d3X: form of M2C3d3 covalently attached in the cell wall-   MES: 2-(N-morpholino)ethanesulphonic acid-   MPLA: monophosphoryl lipid A-   NBT: nitro blue tetrazolium-   OmpA-ss: signal sequence of the outer membrane protein A-   PCR: polymerase chain reaction-   SDS-PAGE: sodium dodecylsulfate polyacrylamide gel electrophoresis-   TDM: trehalose dicorynomycolate-   phP: baculovirus polyhedron promoter-   sgp67: secretion signal for the for the baculovirus-   gp67 protein

EXAMPLE

Introduction

This example demonstrates the preparation of various fusion antigensbased on the influenza A virus M2 protein. The M2 fragment was fused tothe amino terminus of various presenting carriers.

Materials and Methods

1. Bacterial Strains and Plasmids

All plasmid constructions, made for expression in Escherichia coli, wereperformed in strain MC 1061 (hsdR mcrB araD139Δ(araABC-leu)7697 ΔlacX74galU galK rpsL thi (Casadaban and Cohen, 1980) because of highefficiency of transformation. The first transformation after mutagenesiswas performed in WK6λmutS (Δ(lac-proAB), galE, strA,mutS::Tn10/lacI^(q), ZΔM15, proA⁺B⁺; Zell and Fritz, 1987). Expressionstudies of human β₂-microglobulin and derivatives were performed in E.coli strain C3000 (Hfr, sup⁻, thi(λ⁻)). Expression studies of theHepatitis B core protein and derivatives were carried out in MC1061[pcI857].

pcI857 was described in Remaut et al., 1983b. A derivative of thisplasmid pcI857K1 was described in Steidler et al., 1994.

The plasmid p714 (Parker and Wiley, 1989) was a kind gift of Dr. K.Parker and the plasmid pPLcA1 (Nassal, 1988) of Dr. M. Nassal. Theplasmid pPLc245 was described in Remaut et al., 1983a.

For the constructions and expressions in Lactococcus lactis strainMG1363 (Gasson, 1983) was used. The vector for constitutive expressionin L. lactis, pTREX1 (Wells and Schofield, 1996) was a generous giftfrom Dr. K. Schofield. The plasmid pL2MIL2, for the expression ofinterleukin 2, is described in Steidler et al., 1995. An analogousplasmid for the expression of interleukin 6, pL2MIL6, is described inSteidler et al., 1996.

The vector pSG5.C3d.YL (Dempsey et al., 1996) is a gift from Dr. Fearon.

2. Virus

A/PR/8/34 (H1N1) was adapted to mice by several lung passages. Afteradaptation, the virus was grown in eggs (Kendal et al, 1982) andpurified over a sucrose gradient. The titer (haemagglutination units(HAU) (Hirst, 1941; Kendal et al, 1982) and the lethality in mice weredetermined. For m. a. A/PR/8/34, 1 LD₅₀ corresponded to 10 HAU presentin 50 μl.

Influenza strain X-47 (H3N2) (Baez et al., 1980) was used in experimentsfor heterologous challenge. This strain was adapted to mice by severallung passages.

3. Animals

Female Balb/c mice were purchased from Charles River Wiga (Sulzfeld,Germany). The mice were used at the age of 6 to 7 weeks.

4. Antibodies

The monoclonal mouse antibody directed to the Hepatitis B core proteinwas a kind gift from Dr. Sc. H. Claeys (Bloedtransfusiecentrum, Leuven).

A mouse monoclonal antibody specific for the human β₂-microglobulin waspurchased from Boehringer (Mannheim, Germany).

Alkaline phosphatase conjugated antibodies specific for mouse IgG ormouse IgG (γ chain specific) were bought from Sigma Chemical Co. (St.Louis, Mo., USA).

5. Growth Media

E. coli was grown in LB medium (1% tryptone, 0.5% yeast extract and 0.5%NaCl) unless mentioned otherwise. The minimal M9 medium (Miller, 1972),supplemented with 0.2% casamino acids, was used in experiments when theexpressed proteins were secreted into the growth medium and had to bepurified.

M17 growth medium (Difco Laboratories, Detroit, Mich., USA))supplemented with 0.5% glucose (GM 17) was used for culturing L. lactis.Erythromycin was used at a concentration of 5 μg/ml. L. lactis is grownat 28° C. without shaking.

The hybridomas and the myeloma cells were grown in RPMI 1640 (Gibco BRL,Bethesda, Md., USA) supplemented with 10% fetal calf serum, 0.3 mg/mlL-glutamine, 0.4 mM sodium pyruvate, 100 μl/ml penicillin and 100 ng/mlstreptomycin.

5. Adjuvants

For the first immunization Ribi adjuvant (Ribi Immunochem Research Inc.,Hamilton, Mont., USA) was used. A complete dose of Ribi adjuvantcontains 50 μg MPLA (monophosphoryl lipid A), 50 μg TDM (trehalosedicorynomycolate), 2% squalene and 0.01% Tween 80. For the second andthird immunization MPLA (Ribi Immunochem Research Inc., Hamilton, Mont.,Usa) was used alone or mixed with an equal quantity of adjuvant peptide(Sigma Chemical Co., St. Louis, Mo., USA).

6. DNA Manipulations

Restriction enzymes, DNA polymerases, T4 polynucleotide kinase and T4DNA ligase (Boehringer, Mannheim, Germany; Gibco BRL, Bethesda, Md. USA,or New England Biolabs, Beverly, Mass., USA) were used as recommended bythe manufacturer. For analytical purposes, plasmid DNA was extractedaccording to Birnboim and Doly (1979). For preparative purposes, plasmidDNA was isolated according to Kahn et al. (1979). Restriction fragmentsof DNA were isolated by the Geneclean method according to Vogelstein andGillespie (1979) and Struhl (1985). The required materials werepurchased from Bio 101 (La Jolla, Calif., USA). For the isolation ofplasmid DNA out of L. lactis, a pretreatment of the bacteria isnecessary to weaken the cell wall. The bacterial pellet was resuspendedin 50 μl TE (10 mM Tris-HCl pH 8-1 mM EDTA). Afterwards another 50 μlTE, supplemented with 10 mg/ml lysozyme (Boehringer, Mannheim, Germany)and 200 μl/ml mutanolysin (Sigma Chemical Co., St. Louis, Mo., USA) wasadded. This mixture was incubated for 10 min at 37° C. and then put onice for 5 min. Further treatments are identical to those used forplasmid isolation out of E. coli.

For all constructions in L. lactis purified plasmid DNA (Qiagen, Hilden,Germany) were used. The DNA fragments were purified from agarose gels byusing Qiaex II (Qiagen, Hilden, Germany).

7. PCR Amplification

All PCR reactions were carried out following a basic protocol. In eachreaction about 50 ng pure template and 50 μmol sense and anti-senseoligonucleotides (Life Technologies, Paisley, UK) were used. Two unitsVent_(R)® DNA polymerase (New England Biolabs, Beverly, Mass., USA) wereadded after heating of the samples to 94° C. The annealing temperature(T_(a)) was set, according to the composition of the primer, at about 7°C. below the melting temperature (T_(m)). In these PCR amplificationsthe best results were obtained at 60° C. The synthesis of hbc and thefusion genes ipm2hbc and im2hbc, was carried out for 45 seconds at 72°C. The synthesis of the sequence, coding for the extracellular part ofthe M2 protein (cm2 and sm2), was left for 20 seconds at 72° C. A totalof thirty amplification rounds were performed. The control reactions didnot contain oligonucleotides. Three different concentration of MgS0₄were used, 2, 3 and 4 mM. The PCR reaction that produced a significantamount of the expected fragment under the most stringent conditions(lowest Mg²⁺ concentration and highest T_(m)) was used for furthercloning.

The C3d3 fragment was amplified from pSG5.C3d.YL with theoligonucleotides C3ds and C3da using Pwo DNA Polymerase (Boehringer,Mannheim, Germany). The annealing temperature was set at 60° C. and thesynthesis was performed for 2 min at 72° C.

8. Ligation

The ligations for L. lactis were performed with Ready-To-Go™ T4 DNALigase (Pharmacia Biotech, Uppsala, Sweden). After incubation for 1 h at20° C., the mixture was extracted with phenol (Life Technologies,Paisley, UK) and chloroform/iso-amyl alcohol (24/1). The DNA wasprecipitated with see-DNA (Amersham International, Buckinghamshire, UK).The complete resuspended pellet was used for electroporation (Wells etal., 1993).

9. Protein Purification Media

All chromatography media were purchased from Pharmacia Biotech(Uppsala,Sweden), except CF11 cellulose, which was purchased fromWhatman International Ltd. (Maidstone, UK).

10. Protein Gel

Protein samples were analysed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) according to Laemmli, 1970. After electrophoresis, theproteins were fixed with 10% trichloroacetic acid and stained with 0.05%Coomassie brilliant blue R-250 in destain. Excess dye was removed byincubating the gel in destain (30% methanol-7% acetic acid). The gel wassoaked in 40% ethanol before it was dried between two sheets ofpermeable cellophane.

11. Western Blot and Dot Blot

For immunological characterisation, proteins were electrophoreticallytransferred from a SDS-PAGE-gel onto a nitrocellulose membrane (porediameter 0.45 μm, Schleicher & Schuell, Dassal, Germany) with a dryblotting apparatus (Plexi-labo, Gent, Belgium). The filter was blockedfor at least 2h in PBS pH 7.4 (14.5 mM phosphate buffer pH 7.4-150 mMNaCl) with 2.5% skim milk powder and 0.1% Triton X-100 (blockingbuffer). Incubation with the primary antibody, diluted in blockingbuffer, was carried out at room temperature for 30 to 60 min. Excess ofunbound antibody was removed by three washings with blocking buffer. Thebound antibodies were detected with an alkaline phosphatase conjugatedantibody of the appropriate specificity. Subsequently, the filter waswashed two times with PBS pH 7.4-0.1% Triton X-100. A third washing stepwas carried out with substrate buffer (100 mM Tris-HCl pH 9.5-100 mMNaCl-5 mM MgCl₂). The filter was then incubated in substrate buffer with165 μg/ml nitro blue tetrazolium (NBT) and 165 μg/ml5-bromo-4-chloro-3-indolylphosphate (BCIP) until a clear signalappeared. The blot was finally washed thoroughly with tap water anddried.

The dot blot analysis was carried out in a similar way as the Westernblot, except that the proteins were not transferred throughelectrophoresis, but by filtering the samples through a nitrocellulosemembrane.

12. ELISA

In every ELISA we used a 0.1% casein solution for blocking and for thedilution of the antibodies used. The stock solution of casein (2.5%) wasprepared as follows: 6.25 g casein powder was dissolved in 200 ml 300 mMNaOH by overnight stirring at 37° C. The pH was adjusted to 7.0 byadding 2N HCl. The final volume was brought to 250 ml (Nunc bulletin no.7, December 1989). Sodium azide (0.02%) was added as a preservative.

Different ELISA's were developed to determine the concentration ofHepatitis B core or human β2-microglobulin fusion protein. Microtiterplates (type II F96 maxisorp Nunc A/S, Roskilde, Denmark) were coatedfor 1.5 h at room temperature or overnight at 4° C. with a 1/2 dilutionseries of samples containing IPM2HBcm or IPM2hB2Mm. On the same plate, a1/2 dilution series of purified HBc or hB2M, respectively, starting from2 μg/ml, was used as a standard. Between every incubation step, theplates were washed twice with tap water and once with PBS, pH 7.4-0.05%Triton X-100, except that after blocking, the plates were not washed.The microtiter plates were blocked with 0.1% casein solution for 2 h atroom temperature or at 4° C. overnight. As primary antibody we usedmouse anti-HBc or mouse anti-hB2M, respectively. The bound antibodieswere detected with an alkaline phosphatase labelled anti-mouse IgG (γchain specific) antibody. The incubation with antibody solution wascarried out at room temperature for 1.5 h. Finally the microtiter plateswere incubated for 1 h with substrate buffer (10% diethanolamine—0.5 mMMgCl₂—0.02% NaN₃ pH 9.8) containing 1 mg/ml p-nitrophenyl phosphate. Theabsorbance was measured at 405 nm and the wave length of 490 nm was usedfor normalization.

13. Preparation of Polyclonal Anti-M2

All mice, which had been immunized with IPM2HBcm and had survived thelethal challenge with m. a. A/PR/8/34 influenza A virus (see results,immunization) were anaesthetized with 250 μl 25 mg/ml tribromoethanol(injected i.p.) and blood samples were taken by heart puncture. Theserum was isolated as described hereinbelow. The crude serum gave a highbackground in Western blot, therefore an IgG fraction was prepared. Thecrude serum was filtered through a 0.45 μm filter (Millipore Millex-HV,Millipore, Bedford, Mass., USA) and diluted 10 times in loading buffer(PBS—10 mM EDTA pH 8). This mixture was loaded on an equilibratedProtein G Sepharose 4 Fast Flow column (φ=1 cm, h=8 cm). The bound IgGmolecules were eluted with 100 mM glycine-HCl pH 2.7. Fractions of 1 mlwere collected in tubes containing 50 μl 1 M Tris-HCl pH 9.5 to bringthe pH to neutral.

The quantity of anti-M2 antibodies in the pooled peak fractions was 2.6μg/ml. This was determined in an ELISA, comparable to the detection ofanti-M2 antibodies in the serum of immunized mice. Mouse monoclonalanti-human β₂-microglobulin (Cymbus Bioscience, Southampton, UK) wasused as a standard.

14. Serum Preparation

Five blood samples were taken from every mouse: the pre-immune serum(a), the serum taken after the first (b), after the second (c) and afterthe third (d) immunization, and the serum taken after challenge (e).This blood was incubated for 30 min at 37° C. The samples were thenplaced on ice for at least 1 hour and centrifuged two times 5 min at16000 g in a microcentrifuge. The serum was isolated. Equal volumes ofsera obtained from different mice were pooled for the analysis ofantibody production.

15. List of Plasmids

15.1 E. Coli

-   pATIPM2 ml: plasmid that contains the uninterrupted m2 gene from    A/PR/8/34-   pIPM2hB2Mm2s2: plasmid for the expression of IPM2hB2Mm, with the    correct amino terminus of M2-   pPLcIPM2HBc: expression plasmid for IPM2HBc, with four amino acids    between the initiating methionine and the amino terminus of M2e-   pPLcIPM2HBcm: expression plasmid for IPM2HBcm, with the correct    amino terminus of M2e. Sequence of M2 is derived from A/PR/8/34-   pPLcIM2HBcm: expression plasmid for IM2HBcm, with the correct amino    terminus of the universal M2    15.2 L. Lactis-   pT1TT: plasmid for the expression of TTFC-   pT1PM2LTT: expression of IPM2TT, with leucine codons adapted for L.    lactis. Sequence of M2e is derived from A/PR/8/34-   pT1PM2LTTIL2: expression of IPM2TT, with adapted leucine codons, in    combination with mIL2-   pT1PM2LTTIL6: plasmid for the expression of IPM2TT, with adapted    leucine codons, in combination with mIL6-   pT1HBc: plasmid for the expression of HBc-   pT1HBcIL2: expression of HBc in combination with mIL2-   pT1HBcIL6: expression of HBc in combination with mIL6-   pT1PM2HBc: plasmid for the expression of IPM2HBcm. Sequence of M2e    is derived from A/PR/8/34-   pT1PM2HBcIL2: expression of IPM2HBcm in combination with mIL2    pT1PM2HBcIL6: expression of IPM2HBcm in combination with mIL6-   pT1M2HBc: plasmid for the expression of IM2HBcm, with the universal    sequence for M2e-   pT1M2HBcIL2: expression of IM2HBcm in combination with mIL2-   pT1M2HBcIL6: expression of IM2HBcm in combination with mIL6-   pT1PM2LHBc: plasmid for the expression of IPM2HBcm, with leucine    codons adapted for L. lactis-   pT1PM2LHBcIL2: expression of IPM2HBcm, with adapted leucine codons,    in combination with mIL2-   pT1PM2LHBcIL6: plasmid for the expression of IPM2HBc, with adapted    leucine codons, in combination with mIL6-   pT1M2LHBc: expression of IM2HBcm, with leucine codons adapted for L.    lactis-   pT1M2LHBcIL2: expression of IM2HBcm, with adapted leucine codons, in    combination with mIL2-   pT1M2LHBcIL6: expression of IM2HBcm, with adapted leucine codons, in    combination with mIL6-   pT1cM2L: plasmid for the expression of the cytoplasmic form of M2e,    with leucine codons adapted for L. lactis-   pT1cM2LC3d: expression of cM2LC3d, with adapted leucine codons-   pT1cM2LC3d3: expression of cM2LC3d3 (with three consecutive C3d    domains), with adapted leucine codons-   pT1sM2LX: plasmid for the expression of the secreted and anchored    form of M2e, with leucine codons adapted for L. lactis-   pT1sM2LC3d: expression of sM2LC3d, with adapted leucine codons-   pT1sM2LC3d3: expression of sM2LC3d3 (with three consecutive C3d    domains), with adapted leucine codons    Experimental Section    1. Construction of pATIPM2m

The RNA segment 7 of the influenza A virus, A/PR/8/34 (H1N1), was clonedby a procedure as described for RNA segment 4 in Min Jou et al., 1980.The resulting plasmid was named pATIPMA and is commercially available(LMBP catalogue 1992, no. 1774).

The mRNA of the M2 protein is not a colinear transcript of RNA segment7. Indeed, an intron of 689 nucleotides had to be removed (Lamb et al.,1981).

In the plasmid pATIPMA, StuI cuts after the first nucleotide of thesecond exon (see FIG. 1 a). This nucleotide was included in thesynthetic oligonucleotides, that were used to code for the first exon.The synthetic first exon, encoding the amino-terminus of the mature M2protein, was designed to contain a single stranded GATC overhang at its5′ end. This allowed us to make the connection to a preceding BamHI sitein the vector pATIPMA and to replace the original first exon.

Furthermore codon usage was optimized for expression in E. coli.

Next, we introduced, by site-directed mutagenesis (Stanssens et al.,1989), a BclI site at the junction between the extracellular part andthe membrane anchoring region of the M2 protein (see FIG. 1 b). Theamino acid sequence of the extracellular part was not changed. Theresulting plasmid, pATIPM2 ml, carries the uninterrupted m2 gene ofA/PR/8/34.

2. Construction of IPM2hB2Mm

Parker and Wiley (1989) expressed human β2-microglobulin in theperiplasm of E. coli by making use of the plasmid p714. This plasmidcontains the coding region for β₂-microglobulin preceded by the signalsequence of the outer membrane protein A of E. coli (OmpA-ss) (see FIG.2 a). The OmpA signal sequence is required for the translocation of theprotein, to which this sequence is fused, to the periplasm. The signalsequence is cleaved off after transport. On plasmid p714, humanβ2-microglobulin is under control of both the lipoprotein (lpp) andlacUV5 promoter. Addition of 1 mM IPTG to a mid-log phase culture leadsto the production of β₂-microglobulin.

The coding sequence of the extracellular part of the M2 protein,isolated as a BamHI-BclI fragment from pATIPM2 ml, was inserted betweenthe signal sequence of ompA and the human β₂-microglobulin (for detailssee FIG. 2 a). Due to the construction, there were 9 additionalnucleotides between the end of the ompa signal sequence and thebeginning of the m2 fragment, which had to be removed (see FIG. 2 b).This was done by looping out mutagenesis according to Nakamaye andEckstein, 1986. As a result, the plasmid pIPM2hB2Mm2s2 was obtained.

3. Localisation of the IPM2hB2Mm

A freshly grown preculture of C3000 containing p714 or pIPM2hB2Mm2s2 wasdiluted 1/100 in LB with ampicillin. As described above, the hb2m andipm2hb2mm genes are under control of the lacUV5 promoter. When thecultures reached a density of about 5.5×10⁸ bacteria/ml, they weredivided in two and one half of each culture was induced with 1 mM IPTG.After 3 h induction, the bacteria were harvested and fractionated. Theperiplasm of the bacteria was isolated by osmotic shock (Neu and Heppel,1965). The remainder of the bacteria was sonicated (Vibra cell, Sonics &Materials Inc., Danbury, Conn., USA) and centrifuged for 10 min at16000g, to isolate the cytoplasm. The different samples were analyzed ona SDS 15% PAGE-gel. Human B2M and the fusion protein IPM2hB2Mm weretransported to the periplasm, whereas the precursors, where the signalsequence was still present, remained associated with the bacteria (datanot shown). Determination of the amino-terminus of the mature IPM2hB2Mm(by courtesy of Dr. J. Vandekerckhove) by automated Edman degradation ona model 470A gas-phase sequencer coupled to a model 120A on-linephenylthiohydantoin amino acid analyser (Applied Biosystems, FosterCity, Calif., USA), demonstrated that the OmpA signal sequence wascorrectly cleaved off.

4. Purification of IPM2hB2Mm

The fusion protein IPM2hB2Mm could efficiently be expressed in theperiplasm of E. coli. Whereas performing an osmotic shock is a criticalprocedure, especially on large volumes, Steidler et al. (1994)previously described an elegant system, based on the controlledexpression of the Kil protein, to release periplasmic proteins in thegrowth medium.

The kil gene is present on a compatible plasmid under the tightlyregulated P_(L) promoter, the leftward promoter of phage λ (Remaut etal, 1981). The plasmid pcI857K1 also carries the temperature sensitiverepressor of the P_(L) promoter, cI857. The fusion protein IPM2hB2Mm issynthesized upon induction with 1 mM IPTG and at the end of theproduction phase, the culture is switched from 28° C. to 42° C. toinduce kil.

A fermentation (BioFlo IV fermentor, New Brunswick Scientific Co.,Edison, N.J., USA) was carried out using the standard inductionprocedure described above. The culture was centrifuged in a contifuge17RS (Heraeus Instruments, Hanau, Germany) at 11000g and the growthmedium was isolated. The sodium chloride concentration of the growthmedium was adjusted to 300 mM and buffered with 20 mM MES(2-(N-morpholino)ethanesulphonic acid), pH 6.5. This solution was loadedon a DEAE Sephacel column (φ=5 cm, h=6.5 cm), equilibrated with 20 mMMES, pH 6.5-300 mM NaCl. Under these conditions IPM2hB2Mm did not bindto the matrix. The ammonium sulfate concentration of the flow throughwas brought to 0.8 M with a 3.8 M (NH₄)₂SO₄ solution, pH 7. The mixturewas loaded on a Phenyl Sepharose column (φ=5 cm, h=17 cm), equilibratedin 20 mM Tris-HCl, pH 7.5 0.8 M (NH₄)₂SO₄. A decreasing ammonium sulfateconcentration gradient starting from 0.8 M and going to 0, did notrelease the bound fusion protein. This was achieved by eluting thecolumn with a pH gradient from 20 mM Tris-HCl, pH 7.5 to 5 mM NaAc, pH5.5. The peak fractions were pooled and diluted ten times in 20 mMdiethylamine (DEA), pH 8.5.

The complete mixture was loaded on a Sepharose Q column (φp=0.8 cm,h=2.3 cm), equilibrated with 20 mM DEA, pH 8.5. The protein was elutedfrom the column with a salt gradient from 0 to 1M. The peak fractionswere pooled and loaded on a Sephacryl S-100 gel filtration column (φ=1.5cm, h 47 cm). Only one peak with the expected molecular weight of about15 kDa was observed. This purified IPM2hB2Mm was used to immunize micefor preparing hybridomas, secreting monoclonal antibodies directedagainst the M2 protein.

5. Production of Monoclonal Antibodies to the M2 Protein

Balb/c mice were immunized three times with 2.5 μg purified IPM2hB2Mm.For the first injection a complete dose of Ribi adjuvant was used. Thesecond and third immunizations were performed in the presence of 50 μgMPLA. The injections were given with an interval of three weeks. Threedays after the last immunization, spleen cells were isolated and fusedwith myeloma cells SP2/0-AG14 using standard protocols (Köhler andMilstein, 1975). Supernatants from different immunoglobulin producingcell clones were tested in ELISA and Western blot for reactivity againstthe other fusion protein IPM2HBcm (described further). The Hepatitis Bcore protein alone was used as a control to eliminate false positiveclones. The isotype of the antibody was determined (Isostrip,Boehringer, Mannheim, Germany). Two different immunoglobulin subtypesthat recognized the extracellular part of the M2 protein were obtained,an IgM and an IgG2a. Especially the IgG2a antibody was used in furtherexperiments.

6. Expression of HBc and IPM2HBcm

Expression of proteins under control of the P_(L) promoter was performedby shifting an exponentially growing culture from 28° C. to 42° C.(Remaut et al., 1981). A saturated preculture of MC1061 [pcI857]containing the plasmid pPLc245 (control), pPLcA1 (carrying the hbc gene)or pPLcIPM2HBcm (containing the fusion gene ipm2hbc) respectively, wasdiluted 1/100 in LB medium (50 μg/ml kanamycin and 100 μg/ml ampicillin)and grown for about 4 h at 28° C. under shaking. When the culturesreached a density of 4.5×10⁸ to 5.5×10⁸ bacteria/ml, they were split,one half was incubated for 4 h at 28° C., the other half was switched to42° C. The bacteria were concentrated by centrifugation (2 min at 16000gin a microcentrifuge).

The culture medium was removed and the bacteria were resuspended in TEbuffer (10 mM Tris-HCl—1 mM EDTA, pH 7.6). The bacteria were opened bysonication (Vibra cell, Sonics & Materials Inc., Danbury, Conn., USA)and the bacterial debris were pelleted for 10 min at 16000 g in amicrocentrifuge. The supernatant was isolated and the pellet wasresuspended in TE buffer. The samples were analysed on a SDS 12.5%PAGE-gel, in a Western blot and on a dot blot.

7. Large Scale Production of IPM2HBcm

The strain MC1061 [pcI857, pPLcIPM2HBcm] was grown in a BioFlo IVfermentor (New Brunswick Scientific Co., Edison, N.J., USA). When theculture reached a density of about 5.5×10⁸ cells/ml, the temperature wasincreased to 42° C. After three hours of induction, the culture wascentrifuged in a contifuge 17RS (Heraeus Instruments, Hanau, Germany) at11,000g. The bacteria were collected and resuspended in a volume (in ml)buffer (50 mM Tris-HCl pH 8-150 mM NaCl-5% glycerol with one proteaseinhibitor cocktail tablet (Complete™; Boehringer, Mannheim, Germany) per25 ml) corresponding to two times the weight (in g) of the pelletedbacteria. This suspension was treated with 1 mg/ml lysozyme (freshlydissolved in 25 mM Tris-HCl pH 8) for half an hour on ice. Subsequently,the bacteria were lysed with 0.2% Triton X-100 in the presence of 25 mMEDTA, pH 8. After 30 min incubation on ice, the lysates were centrifugedfor 1 h in a Sorvall SS-34 rotor (Du Pont Company, Wilmington, Del.,USA) at 48000 g. The supernatant was removed and used for purificationof IPM2HBcm.

8. Immunization with IPM2HBcm

Balb/c mice were injected three times intraperitoneally with purifiedIPM2HBcm in the presence of adjuvant. Control mice received only PBSbuffer, pH 7.4 and adjuvant. For the first immunization half a dose ofRibi adjuvant was used. In the second and third injection we used 25 μgMPLA and 25 μg MDP.

Mice were immunized intranasally three times by applying a light etheranesthesia, after which 50 microliter antigen solution in PBS buffer(containing either 10 microgram IPM2HBcm or IM2HBcm without anyadjuvant) is put in the nostril.

9. Expression in L. Lactis

Single colonies from L. lactis strain MG 1363, containing the plasmidpT1HBc, pT1PM2HBc or pT1M2HBc, respectively or the derivatives with mIL2(pT1HBcIL2, pT1PM2HBcIL2 and pT1M2HBcIL2) or mIL6 (pT1HBcIL6,pT1PM2HBcIL6 and pT1M2HBcIL6), were inoculated in 10 ml GM17E each.MG1363 [pTREX1] was used as control. The bacteria were grown for about16 h at 28° C. The cells were collected by centrifugation at 2000g for20 min (Sorvall 11 RT6000 D). The growth medium was isolated and thebacteria were resuspended in 250 μl TE. Following resuspension, anadditional 250 μl TE supplemented with 10 mg/ml lysozyme and 200 u/mlmutanolysin was added. This mixture was incubated for 10 min at 37° C.and then put on ice for 5 min. Then 500 μl Laemmli sample buffer (100 mMTris-HCl pH 6.8—5% SDS—1.2M β-mercaptoethanol—0.008% bromophenolblue—16% glycerol) was added and the samples were boiled for 5 min. Anequivalent of 1 ml original culture volume, or 10⁹ bacteria was analyzedon a SDS 12.5% PAGE-gel. The production of mIL2 or mIL6 in the culturesupernatant was evaluated in a bio-assay based on the proliferation ofCTLL2-cells (mIL2, Gillis et al., 1978) or the proliferation of a B-cellhybridoma, 7TD1 (mIL6, Van Snick et al., 1986).

Results

1. Construction of IPM2HBcm

The plasmid pPLcA1 (see FIG. 3 a) contains the hepatitis b core (hbc)gene under control of the P_(L) promoter of bacteriophage λ (a gift fromDr. Nassal). The 346 by NcoI-XbaI HBc fragment, isolated from pPLcA1,was inserted into the NcoI and XbaI opened pMa581, a derivative ofpMa58. This plasmid was called pMaHBc. At the 5′ end of the hepatitis Bcore, directly following the start codon, we introduced a BamHI site bysite-directed mutagenesis (Stanssens et al., 1989), correctly positionedin the reading frame of HBc (for details see FIGS. 3 a and b). Theresulting plasmid was named pMaHBcm. The information coding for theextracellular part of the M2 protein was cloned as a 72 by BamHI-BclIfragment, derived from pATIPM2m1, into the BamHI opened pMaHBcm,resulting in the vector pIPM2HBc. The hbc gene in the expression vectorpPLcA1 was then replaced by the 418 by NcoI-XbaI m2hbc fragment,creating pPLcIPM2HBc. Due to the construction, four amino acids extrawere present between the first methionine and the start of theextracellular part of the M2 protein and had to be removed (see FIG. 3c). This was done by looping out mutagenesis (Deng and Nickolov, 1992).The resulting plasmid was named pPLcIPM2HBcm (see FIGS. 3 a and c).

2. Expression of the Fusion Protein

The plasmids pPLc245 (control), pPLcA1 (hbc gene) and pPLcIPM2HBcm(ipm2hbc gene) were transformed to MC1061 [pcI857]. After culture andinduction the bacteria were lysed by sonication. The lysates werecentrifuged and an aliquot of the supernatants was loaded on a SDS 12.5%PAGE-gel (see FIG. 4). The same fractions were also analysed by aWestern blot. Two different monoclonal antibodies were used: an antibodyspecific for the Hepatitis B core protein and a monoclonal antibody(IgG2a) directed against the extracellular part of the M2 protein.

The monoclonal antibody against Hepatitis B core reveals two differentbands (see FIG. 5A), one corresponding to the Hepatitis B core proteinand the other to the fusion protein. The latter protein has a lowermobility, corresponding to the insertion of the extracellular domain ofthe M2 protein. The presence of the M2 fragment was confirmed by usingthe antibody specific for the extracellular part of the M2 protein (seeFIG. 5B).

The N-terminal amino acid sequence of IPM2HBcm was determined (Dr. J.Vandekerckhove) by automated Edman degradation on a model 470A gas-phasesequencer coupled to a model 120A on-line phenylthiohydantoin amino acidanalyzer (Applied Biosystems, Foster City, Calif., USA). This analysisrevealed the N-terminal sequence Ser Leu Leu, which is exactly the sameas the amino terminal sequence of the M2 protein of the influenza Avirus (FIG. 6). The first amino acid, methionine, was removed in E.coli. The amino-terminus of the fusion protein thus corresponds to thatof the wild type M2 protein (table 1; Lamb et al., 1985).

Hepatitis B core, also when expressed in E. coil, spontaneouslyassociates to form particles, indistinguishable from the viral coreparticles circulating in the blood of Hepatitis B infected patients(Cohen and Richmond, 1982). Clarke and co-workers (1987) showed that apeptide inserted at the amino terminus of the Hepatitis B core proteincould be detected at the surface of the particle.

Electron micrographs (Dr. G. Engler) showed that the IPM2HBcm fusionprotein was able to form similar particles. To investigate whether theinsertion of the extracellular part of the M2 protein resulted in thesurface localization of this fragment, soluble fractions, containing HBcor IPM2HBcm, were loaded on a nitrocellulose membrane in a dot blot. Thedot blots were treated with a monoclonal antibody directed against HBcor against M2. FIG. 7 clearly shows a signal in the soluble pPLcIPM2HBcmfraction, when revealed with the antibody directed against the M2protein (panel B). Since the soluble fraction is loaded in a nativestate onto the nitrocellulose membrane, we conclude that the epitope islocated at the surface of the Hepatitis B core particle.

3. Purification of IPM2HBcm

The bacterial lysates were prepared as described in Materials andmethods. The concentration of Tris-HCl pH 8 and NaCl were adjusted to 20mM and 50 mM respectively. This mixture was loaded on a DEAE Sepharosecolumn (φ=2.5 cm, h=5.5 cm), equilibrated with 20 mM Tris-HCl, pH 8-50mM NaCl. The fusion protein was not retained on the column. To the flowthrough 3.8 M (NH₄)₂S0₄, pH 7 was added to a final concentration of 1.2M. This mixture was incubated under stirring in the cold room during16h. The precipitate was removed over a CF11 cellulose column (φ=2.5 cm,h=3.5 cm). The column was eluted with PBS, pH 7.4. The eluate of about50 ml was concentrated in a Centiprep 30 (Amicon Corporation, Danvers,Ill., USA) to 5 ml and loaded on a Sephacryl S-300 column (φ=2.5 cm,h=91 cm), which was equilibrated with PBS, pH 7.4. The peak fractionswere pooled and the concentration of IPM2HBcm was determined in anELISA. The LPS content was assayed (LAL Coatest® Endotoxin purchasedfrom Endosafe Inc., Charleston, S.C., USA) and was sufficiently low (5to 9 ng/50 μg IPM2HBcm) not to interfere with immunization.

4. Immunization

The purified preparation of IPM2HBcm particles was used to immunize 7weeks old female Balb/c mice. Four different groups of 12 mice wereevaluated. The first group received 50 μg IPM2HBcm, the second 10 μg,the third 5 μg and the fourth a control group, only received buffer withadjuvant. A total of three injections were given with the appropriateadjuvant. The injections were administered with three weeks interval.Three weeks after the last inoculation, the mice were challenged with 5LD₅₀ m.a. A/PR/8/34. The virus was administered intranasally in a totalvolume of 50 μl after ether anesthesia. Morbidity was followed bymeasuring rectal temperature (FIG. 8 A1) and weight (FIG. 8 A2) everyother day.

All mice immunized with IPM2HBcm showed a significant degree ofprotection against the following influenza challenge. Depending on theadministered dose, 9 to 11 mice out of 12 survived the influenzainfection, versus only 2 out of 11 for the control group (see FIG. 8B).

5. Analysis of the Serum Samples

One day prior to the first (bleeding a) and two weeks after everyinjection (bleeding b, c and d) blood samples were taken. Three weeksafter the challenge, when the mice had recovered sufficiently from theinfluenza infection, a last blood sample (e) was taken. The serum wasanalyzed in an ELISA (see Materials and Methods) to identify IgGantibodies directed towards the extracellular part of the M2 protein. Todo so, we made use of the other fusion protein, IPM2hB2Mm. One half ofthe microtiter plate was coated with human β₂-microglobulin, the otherhalf was coated with the fusion protein IPM2hB2Mm, both as unpurifiedculture supernatant. The concentration of IPM2hB2Mm used was 1 μg/ml.The same concentration of total protein was used in both set ups.Therefore, the hB2M content of the culture supernatant of bacteriaexpressing hB2M had to be adjusted to 1 μg/ml by adding purified hB2M(Sigma Chemical Co., St. Louis, Mo., USA). Dilution series (1/3) of thedifferent serum samples, starting from 1/50, were loaded on the hB2M andIPM2hB2Mm, coated wells. The ELISA was further developed as described inMaterials and methods.

To obtain the value for the specific reactivity towards theextracellular part of the M2 protein, the absorbance of hB2M at a givendilution was subtracted from the absorbance of IPM2hB2Mm of thecorresponding dilution. FIG. 9 clearly demonstrates a high antibodyresponse to the extracellular part of the M2 protein, in the mice whichreceived three injections with the vaccine. The titer in the serum wasfurther increased after the challenge.

6. Construction of IM2HBcm

It is the aim of the present invention to make a universal vaccineagainst influenza A viruses. In the vaccination studies described above,we showed protection against the influenza virus from which the originalM2 sequence was derived, A/PR/8/34 (homologous protection). Theextracellular part of the M2 protein from this virus differs from mostother viruses sequenced to date, by only one amino acid (see table 1).Therefore, a construct was made in which the glycine at position 20 waschanged to aspartic acid.

To do so we made use of an intermediate vector in the construction ofpPLcIPM2HBcm, pMaIPM2HBc2 (see Figure). The plasmid pMaIPM2HBc2 does notyet contain the mutated m2 (deletion of 12 extra nucleotides) fragment,which starts at the first mature codon of the M2 protein. Therefore thisfragment was isolated from pPLcIPM2HBcm by cutting with SgrAI and EcoRI.This 499 by SgrAI-EcoRI fragment was cloned into the SgrAI and EcoRIopened vector pMaIPM2HBc2, which resulted in the construction ofpMaIPM2HBc3 (see FIG. 10).

By site-directed mutagenesis according to Deng and Nickoloff (1992) thesequence of the extracellular part of the M2 protein was changed to themore universal M2 sequence (Gly20->Asp). The new plasmid was calledpIM2HBcm. The sequence was determined on a model 373A sequencer (AppliedBiosystems, Foster city, Calif., USA) and shown to contain the desiredmutation. The mutated m2 fragment was isolated from pIM2HBcm as a 499 bySgrAI-EcoRI fragment and reintroduced into the expression vectorpPLcIPM2HBcm, opened with SgrAI and EcoRI, to create pPLcIM2HBcm.

7. Expression of IM2HBcm

Strain MC1061 [pcI857] containing respectively pPLc245, pPLcA1,pPLcIPM2HBcm or pPLcIM2HBcm was cultured as described in theExperimental Section. The bacteria were collected and opened bysonication. The soluble fraction was isolated and the concentration ofHepatitis B core protein or the derived fusion proteins was determinedin an ELISA. A soluble fraction containing 5 μg HBc or I(P)M2HBcm wasanalysed on a SDS 12.5% PAGE-gel (see FIG. 11). The same fractions werealso analysed in a Western blot (see FIG. 12). The proteins of interestwere detected with an antibody directed against the Hepatitis B coreprotein or with the monoclonal antibody specific for the extracellularpart of the M2 protein. It can be concluded that the new fusion protein,IM2HBcm, is expressed as efficiently as IPM2HBcm. Moreover the aminoacid change in the extracellular part of the M2 protein (Gly20-->Asp)has no effect on the binding of the monoclonal anti-M2 antibody.

8. Immunization Against Heterologous Challenge

A similar procedure as described in point 4 was used to test theefficiency of IPM2HBcm and IM2HBcm to protect mice versus heterologouschallenge with influenza. 10 M1crograms of IPM2HBcm or IM2HBcm (purifiedin an identical way as IPM2HBcm) were used for immunization. The micewere challenged with 30 HAU X-47.

All mice immunized showed a significant degree of protection against theheterologous challenge. 8 (in case of IPM2HBcm, p<0.05) or 12 (in caseof IM2HBcm, p<0.0001) mice out of 12 survived the influenza infection,versus only 2 out of 11 in the control group (FIG. 8C).

To test the effect of intranasal administration, the same procedure wasfollowed, but instead of the intraperitoneal injection, the antigen wasadministered intranasally. Also in this case, the protection is evident:12 (in case of IPM2HBcm, p<0.0001) or 11 (in case of IM2HBcm, p<0.001)mice out of 12 survived the influenza infection, versus 2 out of 11 inthe control group (FIG. 8D).

9. Construction of Vectors for the Expression of M2-HBc Fusion Proteinsin L. Lactis

The plasmid pTREX1 (Wells and Schofield, 1996) was used to express theHepatitis B core protein and two M2-HBc fusion proteins, IPM2HBcm andIM2HBcm, in Lactococcus. This plasmid has a constitutive L. lactischromosomal promoter, P1, which is followed by the translationinitiation region of the E. coli bacteriophage T7 gene 10 (Wells andSchofield, 1996). The transcription terminator is derived from T7 RNApolymerase. The plasmid pTREX1 also carries two genes for resistance toerythromycin.

The expression plasmid, pTREX1, was cut with SphI, leaving a 3′CATGextension which was removed with Klenow DNA polymerase. The removednucleotides were included in the sense linker for PCR amplification ofthe different genes. The linearized vector was then cut with BamHI andtreated with CIP (calf intestine phosphatase, Boehringer, Mannheim,Germany).

The genes hbc, ipm2hbc and im2hbc were amplified by PCR (see Materialsand methods). The anti-sense linker (HBca) was identical in allamplifications and provided a SpeI and a BclI site after the stop codon(see FIG. 13). For the amplification of ipm2hbc and im2hbc the samesense oligonucleotide (M2s) could be used, since the mutation Gly→Asp inthe extracellular part of the M2 protein is located further downstream.

The amplification of hbc from pPLcA1 was only possible after the vectorhad been linearized with ScaI. The amplification reaction that produceda sufficient amount of fragment, under the most stringent conditions,was used for further cloning. The amplified fragment, hbc, ipm2hbc orim2hbc, was cut with BclI, phosphorylated with T4 polynucleotide kinaseand inserted in the SphI and BamHI opened pTREX1 (see FIG. 14). The newplasmids were called pT1HBc, pT1PM2HBc (in which the extracellular partof the M2 protein is derived from the virus A/PR/8/34) and pT1M2HBc (inwhich the sequence of the extracellular part of the M2 proteincorresponds to the type present in nearly all human influenza A virusessequenced to date), respectively. The sequence of the inserted fragmentwas determined on a model 373A sequencer (Applied Biosystems, FosterCity, Calif., USA) and shown to be correct.

In view of using Lactococcus lactis as an improved vaccine deliveryvehicle, two murine cytokines, interleukin 2 (mIL2) and interleukin 6(mIL6) were inserted as second cistrons in the same operon as theantigen. In that way we could obtain bacteria expressing the antigen,e.g. IM2HBcm, together with secreted murine interleukin 2 or 6. Toobtain secretion of the interleukins into the growth medium, they werefused in frame to the lactococcal usp45 secretion signal peptide (vanAsseldonk et al., 1990). The plasmids pT1HBc, pT1PM2HBc and pT1M2HBcwere cut with SpeI and treated with CIP. The murine interleukin 2 genewas isolated as a 572 by XbaI-SpeI fragment from plasmid pL2MIL2(Steidler et al., 1995). This fragment was inserted into the SpeI openedpT1HBc, pT1PM2HBc and pT1M2HBc giving rise to pT1HBcIL2, pT1PM2HBcIL2and pT1M2HBcIL2, respectively.

In an analogous way the murine interleukin 6 gene was isolated as a 687by XbaI-SpeI fragment from pL2MIL6 (Steidler et al., 1996) and insertedinto the SpeI opened vectors, pT1HBc, pT1PM2HBc and pT1M2HBc, to createpT1HBcIL6, pT1PM2HBcIL6 and pT1M2HBcIL6, respectively.

10. Expression of HBc and M2HBc in L. Lactis

Lactococcus lactis strain MG1363 (Gasson, 1983) containing the plasmidsfor the expression of the antigen alone (pT1HBc, pT1PM2HBc and pT1M2HBc)or in combination with mouse interleukin 2 (pT1HBcIL2, pT1PM2HBcIL2 andpT1M2HBcIL2) or mouse interleukin 6 (pT1HBcIL6, pT1PM2HBcIL6 andpT1M2HBcIL6) were cultured as described in Materials and methods. MG1363[pTREX1] was used as control.

An equivalent of 10⁹ bacteria was analysed by SDS 12.5% PAGE. Theexpression of the Hepatitis B core and the M2-HBc fusion proteins wereanalysed by Western immunoblotting (see FIG. 15) carried out asdescribed in Materials and methods. The expression of IM2HBc in MG1363[pT1M2HBcIL6] was not as high as in the other constructs. By screeningdifferent colonies a clone could be isolated with comparable expressionlevels.

The production and secretion of interleukins into the growth medium wasanalyzed in a biological assay. The biological activity of mIL2 wasassayed by the proliferation of a T-cell line, CTLL2 (Gillis et al.,1978) as compared to a human IL2 standard. The biological activity ofmIL6 was measured by the proliferation of a B-cell hybridoma, 7TD1 (VanSnick et al., 1986). Table 2 gives an overview of the level ofinterleukin 2 and 6 per ml culture medium produced by the differentexpression plasmids. The supernatant of cultures producing mIL6 did notlead to proliferation in a mIL2 assay and vice versa.

TABLE 2 Plasmid mIL2 production mIL6 production pT1HBcIL2 410 ng/ml —pT1PM2HBcIL2 481 ng/ml — pT1M2HBcIL2 359 ng/ml — pT1HBcIL6 — 1020 ng/ml pT1PM2HBcIL6 — 772 ng/ml pT1M2HBcIL6 — 802 ng/ml11. Adaptation of the Coding Sequence of M2e to Expression in L. Lactis

Since the two fusion proteins, IPM2HBcm and IM2HBcm could hardly bedetected in a Western blot, we tried to augment the production of thesetwo fusion proteins by adapting the codon usage of the extracellularpart of the M2 protein to L. lactis (van de Guchte et al., 1992).

At the 5′ end of the extracellular part of the M2 protein we observedtwo consecutive leucine codons (CUG CUG) that were optimal forexpression in E coli (68%), but poor for translation in L. lactis (8%,percentages described in van de Guchte et al., 1992). Therefore thesecodons were changed to UUA. The genes for ipm2hbc and im2hbc wereamplified by PCR from respectively pPLcIPM2HBcm or pPLcIM2HBcm, with anew sense primer, M2Ls, containing the two changed leucine codons (seeFIG. 13). As anti-sense primer we used again HBca (see FIG. 13). Thecloning of the genes was analogous as depicted in FIG. 14. The vectorsso created were called pT1PM2LHBc and pT1M2LHBc.

The expression level of the mutated M2HBc proteins, compared to theoriginal fusion proteins, was analyzed in a Western blot (see FIG. 16).The expression level of the M2HBc fusion proteins with the L. lactisadapted leucine codons, was indeed much higher. It is concluded that theadaptation of codon usage to the L. lactis translation machinery, has apositive effect on the level of protein produced. In a similar way asdescribed above, the murine interleukin 6 gene was inserted intopT1PM2LHBc and pT1M2LHBc, giving rise to pT1PM2LHBcIL6 and pT1M2LHBcIL6,respectively.

12. Construction of M2C3d in Lactococcus Lactis

A second carrier protein, C3d, is also an attractive molecule for thepresentation of the extracellular part of the M2 protein. Dempsey et al.(1996) demonstrated that the attachment of an antigen to threeconsecutive C3d molecules, was much more efficient in producing a highantibody response than the antigen administered in complete Freund'sadjuvant.

The universal sequence of the extracellular part of the M2 protein, withthe adapted leucine codons, is used for making a fusion to theamino-terminus of the first C3d molecule. The coding sequence for threedifferent fusion proteins are constructed. In the first situation theM2C3d3 fusion protein is expressed in the cytoplasm of L. lactis(cM2C3d3), similar to the M2HBc fusion proteins. In the second case theM2C3d3 protein is secreted into the growth medium by making an in framefusion to the usp45-signal sequence (sM2C3d3) and the last construct,which is a derivative of the secreted form, contains in addition ananchor sequence (spaX) after the last C3d molecule to attach the fusionprotein covalently in the cell wall (sM2C3d3X).

The amplified C3d3 fragment was first subcloned in a derivative ofpUC18, namely pUCB/S. pUC18 was linearized with HindIII and a BglIIlinker was inserted. The resulting plasmid was then opened with SmaI anda SpeI linker was inserted, resulting in the plasmid pUCB/S (see FIG.18). Three succeeding copies of C3d were amplified from pSG5.C3d3.YL (agift from Dr. D. Fearon) by PCR with the oligonucleotides C3ds and C3da(see FIG. 17). This amplified fragment was cut with BglII and SpeI. Theresulting 2830 by BglII-SpeI fragment was cloned into the BglII and SpeIopened vector pUCB/S (see FIG. 18). The genes cm2 and sm2 were amplifiedby PCR. For the amplification of cm2 we used the sense oligonucleotideM2Ls (see FIG. 13) and the anti-sense linker M2Ca, which carried for ourpurposes a BamHI site in the correct reading frame (see FIG. 17). Thesame anti-sense linker was used for the amplification of sm2. The senseoligonucleotide for the amplification of sm2, M2LSs, started at thefirst codon of the mature M2 protein.

For the synthesis of the cytoplasmic form of M2C3d3, the informationcoding for the extracellular part of the M2 protein was inserted intopTREX1 analogous as the m2hbc gene described above (see also FIG. 18).The amplified cm2 fragment was cut with BamH I (77 bp), phosphorylatedwith T4 polynucleotide kinase and inserted in the Sph I and BamH Iopened pTREX1, creating pT1cM2L. For the synthesis of the secreted andanchored form of M2C3d3, the information coding for the extracellularpart of the M2 protein was inserted into pT1NX. The vector pT1NX carriesthe usp45-signal sequence (usp45-ss) and the anchor sequence derivedfrom Staphylococcus aureus protein A (spaX). The plasmid pT1NX was cutwith Nae I, correctly positioned at the end of the usp45-ss and BamH I.The amplified fragment, sm2, was cut with BamH I and phosphorylated withT4 polynucleotide kinase. This 73 by sm2 fragment was inserted into theNae I and BamH I opened pT1NX, resulting in the plasmid pT1sM2LX (seeFIG. 18). One single C3d fragment, isolated from pUCC3d, can then beinserted into the BamH I site at the end of the cm2 or sm2 sequence.Afterwards one or two additional C3d copies will be inserted.

13. Construction of M2TTFC in Lactococcus lactis

A third carrier protein, tetanus toxin fragment C (TTFC), can also beused. TTFC has already been expressed in L. lactis under control of theP1 promoter, pT1TT (Wells and Schofield, 1996). L. lactis expressingTTFC in combination with mIL2 or mIL6 to raise the antibody production,was successfully used in immunization experiments (Patent GB 9521568.7).As positive control for analysis of antibody response in the presentimmunization experiments with L. lactis expressing I(P)M2HBcm, a fusionwas made between the extracellular part of the M2 protein and the aminoterminus of TTFC.

The ttfc gene was amplified by PCR (see Materials and Methods) frompT1TT. The sense oligonucleotide (TTFCs) provided a BamH I site,positioned in the correct reading frame, before the second codon ofttfc, corresponding to threonine. The anti-sense linker (TTFCa) provideda Spe I and a BamH I site after the stop codon (see FIG. 19). Theamplification reaction that produced a sufficient amount of fragment,under the most stringent conditions, was used for further cloning (seeMaterials and Methods). The amplified ttfc fragment was cut with BamH I,phosphorylated with T4 polynucleotide kinase and inserted in the Bcl Iopened pATIPM2 ml (see FIG. 20). This plasmid construct was calledpATIPM2TT. From this plasmid the m2ttfc gene was amplified by PCR (seeMaterials and methods) with M2Ls and TTFCa (see FIG. 19). The amplifiedm2ttfc fragment was cut with BamH I, phosphorylated with T4polynucleotide kinase and inserted in the Sph I and BamH I opened pTREX1(see FIG. 20). The new plasmid was called, pT1PM2LTT. In this constructthe extracellular part of the M2 protein is derived from the virusA/PR/8/34, with the two leucine codons adapted for use in L. lactis. Thesequence of the inserted fragment was determined on a model 373Asequencer (Applied Biosystems, Foster City, Calif., USA) and shown to becorrect.

The murine interleukin genes, mIL2 and mIL6, were inserted in the sameoperon as m2ttfc. The murine interleukin 2 gene was isolated as a 572 byXba I—Spe I fragment from plasmid pL2MIL2 (Steidler et al., 1995). Thisfragment was inserted into the Spe I opened pT1PM2LTT giving rise topT1PM2LTTIL2 (see FIG. 20). In an analogous way the murine interleukin 6gene was isolated as a 687 by Xba I—Spe I fragment from pL2MIL6(Steidler et al., 1996) and inserted into the Spe I opened vectorpT1PM2LTT to create pT1PM2LTTIL6 (see FIG. 20).

14. Expression of TTFC and M2TTFC in L. Lactis

Lactococcus lactis strain MG1363 (Gasson, 1983) containing the plasmidsfor the expression of the antigen alone (pT1PM2LTT) or in combinationwith mouse interleukin 2 (pT1PM2LTTIL2) or mouse interleukin 6(pT1PM2LTTIL6) were cultured as described in Materials and methods.MG1363 [pT1TT] was used as a control.

An equivalent of 10⁹ bacteria was analyzed by SDS 10% PAGE. Theexpression of the IPM2TTFC fusion protein was analyzed by Westernimmunoblotting (see FIG. 21) carried out as described in Materials andmethods.

The production and secretion of interleukins into the growth medium wasanalyzed by a biological assay. L. lactis [pT1PM2LTTIL2] produced about500 ng/ml mIL2 and L. lactis [pT1PM2LTTIL6] about 1 pg/ml mIL6. Theseresults are comparable with the expression levels obtained withI(P)M2HBcm in combination with the two interleukins.

Discussion

The present example describes several systems for the presentation ofthe highly conserved extracellular part of the influenza A virus M2protein to the immune system. The M2 fragment was fused to the aminoterminus of the carrier protein in order to retain a free N-terminus ofthe M2-domain and in this way mimic the wild type structure of the M2protein. The first fusion protein, M2 linked to human β₂-microglobulin(IPM2hB2Mm), was used to produce monoclonal antibodies. A second fusionprotein, M2 linked to Hepatitis B core protein (IPM2HBcm) was used forvaccination studies. Both proteins could also be used in the detectionof a specific antibody response against the extracellular part of the M2protein, since a correction has to be made for antibodies directedagainst the carrier protein, which are also produced during theimmunization process.

The vaccination studies with IPM2HBcm showed that the administered dosein the range that was used, was apparently not a very critical parameterfor obtaining protection, as a dose ranging from 5 to 50 μg protectedthe mice, although the immunized mice still showed a high morbidity.This may have been due to the high dose of virus (5 LD₅₀) that was usedfor the challenge in order to obtain a clear-cut result for the degreeof protection. In a natural influenza infection the number of infectingvirus particles is much lower, so that it can be assumed that themorbidity would decrease accordingly.

Analysis of the serum of immunized mice showed a substantial antibodyresponse towards the extracellular part of the M2 protein, especiallyafter viral challenge. This latter, high response can be due to anotherway of administration, intraperitoneal versus intranasal. Or it can beexplained on the basis of a more complete defense mechanism against theincoming virus.

Slepushkin et al. (1995) described a vaccination strategy, based on amembrane extract containing the natural complete M2 protein forhomologous and heterologous virus challenge. But they used a very strongadjuvant, incomplete Freund's, which is not appropriate for medical use.

In contrast, the M2 extracellular domain fusions of the inventiondescribed here can be obtained in a pure form (at least 95% purity), andcan be administered in combination with safe adjuvants. A high degree ofprotection was obtained, despite the fact that the challenge was fairlysevere. In view of the almost invariant sequence of the M2 extracellulardomain (see table 1 which shows an overview of the amino acid sequencesof the extracellular domain of the influenza A M2 protein) it may beexpected that the protection achieved will be similar against all humaninfluenza A strains known so far.

The vaccine may be further improved by the inclusion of an influenzaspecific T helper epitope as well as a CTL epitope into the fusionprotein, for example internally or linked to the C-terminus of theHepatitis B core protein. Other immunization routes are possible aswell, for example intraperitoneal versus intranasal.

Besides the gram negative organism, E. coli, also L. lactis was used, agram positive organism, for the expression of the M2HBcm fusionproteins. In L. lactis it is not necessary to purify the expressedfusion protein. The bacteria can be administered directly eitherintranasally or orally.

A third promising carrier protein is also described, namely the thirdcomplement protein fragment d (C3d) (Dempsey et al., 1996). In apreferred construction, three copies of the C3d protein are preceded bythe extracellular domain of the M2 protein. This M2C3d3 fusion proteincan be expressed either intracellular, anchored in the cell wall orsecreted into the growth medium by genetic fusion to appropriateregulatory sequences.

REFERENCES

-   Allen et al. (1980) Virology 107, 548-551-   Baez et al. (1980) J. Infect. dis. 141, 362-365-   Belshe et al. (1988) J. Virol. 62, 1508-1512-   Birnboim and Doly (1979) N.A.R. 7, 1513-1523-   Borisova et al. (1989) FEBS Lett. 259, 121-124-   Casadaban and Cohen (1980) J. Mol. Biol. 0.138, 179-207-   Clarke et al. (1987) Nature 330, 381-384-   Cohen and Richmond (1982) Nature 296, 677-678-   Cox et al. (1988) Virology 167, 554-567-   Dempsey et al. (1996) Science 271, 348-350-   Deng and Nickolov (1992) Anal. Biochem. 200, 81-88-   Gasson (1983) J. Bact. 154, 1-9-   Gillis et al. (1978) J. Immunol. 120, 2027-2032-   Hirst (1941) Science 94, 22-23-   Holsinger and Lamb (1991) Virology 183, 32-43-   Kahn et al. (1979) Methods Enzymol. 68, 268-280-   Kendal et al. (1982) Concepts and procedures for laboratory-based    influenza surveillance. p. B7-Bl 2, Bl 7-Bl 9-   Klimov et al. (1992) Virology 186, 795-797-   Köhler and Milstein (1975) Nature 256, 495-497-   Laemmli (1970) Nature 227, 680-685-   Lamb and Lai (1981) Virology 112, 746-751-   Lamb et al. (1981) Proc. Natl. Acad. Sci. USA 78, 4170-4174-   Lamb et al. (1985) Cell 40, 627-633-   Levi and Amon (1996) Vaccine 14, 85-92-   Markushin et al. (1988) Virus Res. 10, 263-272-   Miller (1972) Experiments in Molecular Genetics. Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y., p. 431-   Min Jou et al. (1980) Cell 19, 683-696-   Nakamaye and Eckstein (1986) N.A.R. 14, 9679-9698-   Nassal (1988) Gene 66, 279-294-   Neu and Heppel (1965) J. Biol. Chem. 240, 3685-3692-   Ortin et al. (1983) Gene 23, 233-239-   Parker and Wiley (1989) Gene 83, 117-124-   Remaut et al. (1981) Gene 15, 81-93-   Remaut et al. (1983a) N.A.R. 11, 4677-4688-   Remaut et al. (1983b) Gene 22, 103-113-   Schader et al. (1992) J. Virol. 66, 106-114-   Slepushkin et al. (1995) Vaccine 13, 1399-1402-   Stanssens et al. (1989) N.A. R. 17, 4441-4454-   Steidler et al. (1994) Biotechn. Bioeng. 44, 1074-   Steidler et al. (1995) Appl. Environ. Microbiol. 61, 1627-1629-   Steidler et al. (1996) NATO ASI Series H 98 p 63-79. eds.    Bozoglu, T. F. and Ray, B. Springer, Berlin-   Struhl (1985) Biotechniques 3, 452-453-   Sugrue et al. (1990) Virology 179, 51-56-   Sugrue and Hay (1991) Virology 180, 617-624-   Treanor et al. (1990) J. Virol. 64, 1375-1377-   van Asseldonk et al. (1990) Gene 95, 155-160-   van de Guchte et al. (1992) FEMS Microbiol. Rev. 88, 73-92-   Van Snick et al. (1986) Proc. Natl. Acad. Sci. USA 83, 9679-   Vogelstein and Gillespie (1979) Proc. Natl. Acad. Sci. USA 76,    615-619-   Wells et al. (1993) J. Appl. Bact. 74, 629-636-   Wells and Schofield (1996) NATO ASi Series H 98 p 37-62. eds.    Bozoglu, T. F. and Ray, B. Springer, Berlin-   Winter and Fields (1980) N.A.R. 8, 1965-1974-   Zebedee and Lamb (1988) J. Virol. 62, 2762-2772-   Zebedee and Lamb (1989) N.A.R. 17, 2870-   Zell and Fritz (1987) EMBO J. 6, 1809-1815

1. A human influenza immunogenic composition comprising a fusionproduct, said fusion product comprising (i) an antigen comprising animmunogenic extracellular part of an M2 membrane protein of a humaninfluenza A virus, wherein said extracellular immunogenic part consistsof SEQ ID NOs: 1, 2, or 3, or an immunogenic fragment thereof thatinduces antibodies to human influenza A virus, and (ii) a presentingcarrier.
 2. The composition of claim 1, wherein the presenting carrieris heterologous peptide or polypeptide.
 3. The composition of claim 2,wherein the heterologous peptide or polypeptide is selected from thegroup consisting of a hepatitis B core protein, C3d, a polypeptidecomprising multiple copies of C3d, tetanus toxin fragment C and yeast Typarticles.
 4. The composition of claim 1, wherein the presenting carrieris a non-peptidic structure.
 5. The composition of claim 4, wherein thepresenting non-peptidic structure is selected from the group consistingof glycans, polyethylene glycols, peptide mimetics, and syntheticpolymers.
 6. The composition of claim 1, wherein the presenting carrierenhances the immunogenicity of the antigen.
 7. The composition of claim6, wherein the presenting carrier comprises an epitope recognized by aninfluenza-specific T helper cell or cytotoxic T cell.
 8. The compositionof claim 1, wherein the composition comprises Lactococci cellsexpressing said fusion product in or on their cell membrane, and saidcells can release said fusion product.
 9. The composition of claim 1,wherein the fusion product is in an isolated form.
 10. The compositionof claim 1, wherein the fusion product is anchored in the membrane of anacceptor cell expressing the fusion product.
 11. The composition ofclaim 1, wherein the fusion product is part of a lipid bilayer or cellwall.
 12. The composition of claim 1, wherein the composition comprisesLactococci cells expressing said fusion product in or on their cellwall.
 13. The composition of claim 1, further comprising an influenzaantigen selected from the group consisting of hemagglutinin,neuraminidase, nucleoprotein and native M2.
 14. A method of obtaining ahuman influenza immunogenic composition, comprising providing a fusionproduct, said fusion product comprising (i) an antigen comprising animmunogenic extracellular part of an M2 membrane protein of a humaninfluenza A virus, wherein said extracellular immunogenic part consistsof SEQ ID NOs: 1, 2, or 3, or an immunogenic fragment thereof thatinduces antibodies to human influenza A virus, and (ii) a presentingcarrier; and mixing said fusion product with an excipient.
 15. Themethod according to claim 14, wherein the presenting carrier isheterologous peptide or polypeptide.
 16. The method according to claim14, wherein the heterologous peptide or polypeptide is selected from thegroup consisting of a hepatitis B core protein, C3d, a polypeptidecomprising multiple copies of C3d, tetanus toxin fragment C and yeast Typarticles.
 17. The method according to claim 14, wherein the presentingcarrier is a non-peptidic structure.
 18. The method according to claim14, wherein the presenting non-peptidic structure is selected from thegroup consisting of glycans, polyethylene glycols, peptide mimetics, andsynthetic polymers.
 19. A human influenza immunogenic compositionobtained by the following steps: providing a nucleic acid construct thatencodes a fusion product, said fusion product comprising (i) an antigencomprising an immunogenic extracellular part of an M2 membrane proteinof a human influenza A virus, wherein said extracellular immunogenicpart consists of SEQ ID NOs: 1, 2, or 3, or an immunogenic fragmentthereof that induces antibodies to human influenza A virus, and (ii) aheterologous peptide or polypeptide presenting carrier; introducing thenucleic acid construct into an acceptor cell form a transformed acceptorcell; culturing the transformed acceptor cell in a culture medium underconditions that permit expression of the fusion product; and optionallyisolating the fusion product from the acceptor cell or its culturemedium; and optionally admixing the fusion product with an excipient,thereby obtaining a human influenza immunogenic composition.
 20. Theinfluenza immunogenic composition of claim 19, wherein the heterologouspeptide or polypeptide is selected from the group consisting of ahepatitis B core protein, C3d, a polypeptide comprising multiple copiesof C3d, tetanus toxin fragment C and yeast Ty particles.
 21. Theinfluenza immunogenic composition of claim 20, wherein the influenzaimmunogenic composition comprises a cytokine.
 22. The influenzaimmunogenic composition of claim 20, wherein the influenza immunogeniccomposition comprises a vaccine adjuvant that is not Freund's adjuvant.23. An influenza immunogenic composition for an animal speciescomprising a fusion product, said fusion product comprising (i) anantigen comprising an immunogenic extracellular part of an M2 membraneprotein of an influenza A virus, wherein said extracellular immunogenicpart consists of SEQ ID NOs: 1, 2, or 3, or an immunogenic fragmentthereof that induces antibodies to human influenza A virus; and (ii) apresenting carrier.
 24. The composition of claim 23, wherein thepresenting carrier is heterologous peptide or polypeptide.
 25. Thecomposition of claim 24, wherein the heterologous peptide or polypeptideis selected from the group consisting of a hepatitis B core protein,C3d, a polypeptide comprising multiple copies of C3d, tetanus toxinfragment C and yeast Ty particles.
 26. The composition of claim 24,wherein the presenting carrier is a non-peptidic structure.
 27. Thecomposition of claim 25, wherein the presenting non-peptidic structureis selected from the group consisting of glycans, polyethylene glycols,peptide mimetics, and synthetic polymers.
 28. A human influenzaimmunogenic composition comprising a fusion polypeptide, said fusionpolypeptide comprising (i) an antigen comprising an immunogenicextracellular part of an M2 membrane protein of an influenza A virus,wherein said extracellular immunogenic part consists of SEQ ID NOs: 1,2, or 3, or an immunogenic fragment thereof that induces antibodies tohuman influenza A virus; and (ii) a heterologous peptide or polypeptidepresenting carrier, said fusion polypeptide being the expression productof a gene construct comprising a coding sequence for said antigen of (i)linked to a coding sequence for said presenting carrier of (ii).
 29. Theinfluenza immunogenic composition of claim 28, wherein said heterologouspeptide or polypeptide presenting carrier is selected from the groupconsisting of a hepatitis B core protein, C3d, a polypeptide comprisingmultiple copies of C3d, and tetanus toxin fragment C.
 30. The influenzaimmunogenic composition of claim 20, wherein said heterologous peptideor polypeptide presenting carrier is the hepatitis B core protein.